In liver transplant recipients the risk of allograft rejection is greatest in the first few weeks after transplantation, although it can occur as late as the 8th post-operative month. Rejection is most common, however, between the 4th and 10th post-operative days. Prompt diagnosis is crucial to limit damage by this allogeneic immune response. It is also vital that the diagnosis is correct, since administration of augmented immunosuppression in the absence of rejection has its own morbidity--particularly in delaying wound healing and in predisposing the patient to serious infection. The most reliable evidence of rejection is histological but may not always be possible due to severe impairment of clotting. In current practice, the suspected diagnosis of rejection usually rests on evidence of progressive deterioration of liver function in the absence of any other explanation for this functional derangement (Calne, Ry, (1987); Liver Transplantation (2nd Edition), Ed Calne Ry, Grune & Stratton, Inc., London, 301-303). Thus, significant rejection is not diagnosed unless the serum bilirubin and alkaline phosphatase levels are elevated. If the serum transaminases and prothrombin time are also rising, then rejection is assumed unless there is evidence of (a) portal vein/hepatic artery obstruction, (b) septicaemia or (c) drug toxicity. The duration of treatment of rejection by augmented immunosuppression will depend on the improvement in liver function tests (LFTs). Patients with persistently raised LFTs have a poor prognosis.
The biochemical assessment of liver function usually includes measurement of plasma or serum aspartate aminotransferase (AST) or alanine aminotransferase (ALT) activity. These cytosolic enzymes are released into the circulation following hepatocellular damage. The measurement of these aminotransferases for monitoring liver function has been questioned, however, as activities may be normal in patients with chronic liver disease. The poor sensitivity of aminotransferases in detecting damage in certain types of liver pathology may partly lie in their distribution within the liver. The periportal hepatocytes contain the highest concentrations of the aminotransferases but the centrilobular hepatocytes, which are relatively deficient in aminotransferases, are more susceptible to damage from hypoxia and toxins such as alcohol and paracetamol.
Recently, the measurement of hepatic alpha glutathione S-transferase (.alpha.-GST) has been advocated as a superior marker of hepatocellular damage than the aminotransferases in a variety of clinical conditions including halothane hepatotoxicity (Hussey, A. J. et al. (1988); Br. J. Anaesth., 60, 130-135), autoimmune chronic active hepatitis (Hayes, P. C. et al. (1988); Clin. Chem. Acta., 172, 211-216), birth asphyxia (Beckett, G. J. et al. (1989) Clin. Chem. 35, 995-999) and paracetamol poisoning (Beckett, G. J. et al. (1989); Clin. Chem., 35, 2186-2189). Indeed, studies of acute liver damage and chronic active hepatitis have indicated that GST activities, unlike those of transaminase, correlate better with histological abnormalities. (Bass, N. M. et al. (1978); Gastroenterology, 75, 589-594 and Sherman, M. et al. (1983); Hepatology, 3, 162-169).
The GSTs are a complex family of enzymes involved in detoxification. The enzymes catalyze the nucleophilic attack of glutathione (GSH) on a wide range of hydrophobic electrophiles. The GSTs can be divided into three classes: the basic, the near-neutral and the acidic enzymes according to their isoelectric points. These classes are related to the alpha (I), mu (II) and pi (III) families, respectively, in the rat (2). The human alpha class enzymes, also referred to as "ligandin" (3), comprise two subunits B.sub.1 and B.sub.2 which can hybridize to form the homodimers, B.sub.1 B.sub.1 and B.sub.2 B.sub.2 and the heterodimer, B.sub.1 B.sub.2 (4).
Adult liver contains perdominantly the basic or .alpha.-GST. One of the properties of hepatic GST which may partly explain its greater sensitivity as a marker of liver damage when compared with the aminotransferases is its wider distribution within the liver. Immunohistochemical studies of GST in human foetuses, neonates and adults have shown that the basic and acidic GST are equally expressed in both periportal and centrilobular hepatocytes (Hiley C. et al. (1988); Biochem. J., 284, 255-259). Other properties of GSTs also offer theoretical advantages over measurements of aminotransferases in the investigation of liver damage. They are relatively small enzymes (MW.about.50,000) and are present in high concentrations in the hepatocyte cytosol. GSTs are readily and rapidly released in quantity into the circulation following hepatic damage; their short plasma half-life (&lt;90 min.) allowing early detection of hepatic damage and its resolution.
Histologically the acute rejection process appears to begin with a mononuclear cell infiltration of portal tracts. It is noteworthy that spillover of lymphocytes into the adjacent periportal parenchyma is occasionally but not always a feature of acute rejection. There may also be involvement of centrilobular blood vessels and bile ducts. The pattern of infiltration within the liver is also variable--ranging from focal to diffuse. The diagnostic sensitivity of the transaminases as early markers of rejection depends on their distribution within the liver in relation to those sites involved at the beginning of the rejection process. If the periportal hepatocytes are not affected in this early pathological process, changes in transaminases may not always be expected to rise markedly and diagnosis and treatment could be delayed. This may be particularly important in the first few days after transplantation, when the patient is at greatest risk of rejection and transaminases remain elevated as a result of ischaemia and surgical trauma to the liver.
It is an object of the present invention to provide a method which assists in the early diagnosis of rejection in liver transplant recipients.
It is a further object of the present invention to provide a more sensitive and specific marker of the rejection process in the liver transplant recipient than has heretofore been available.
It is a still further object of the present invention to provide a method for the monitoring of post-operative liver transplant patients so that earlier corrective action can be taken which may prevent rejection occurring.